Architecture and regulation of a GDNF-GFRα1 synaptic adhesion assembly

Glial-cell line derived neurotrophic factor (GDNF) bound to its co-receptor GFRα1 stimulates the RET receptor tyrosine kinase, promoting neuronal survival and neuroprotection. The GDNF-GFRα1 complex also supports synaptic cell adhesion independently of RET. Here, we describe the structure of a decameric GDNF-GFRα1 assembly determined by crystallography and electron microscopy, revealing two GFRα1 pentamers bridged by five GDNF dimers. We reconsitituted the assembly between adhering liposomes and used cryo-electron tomography to visualize how the complex fulfils its membrane adhesion function. The GFRα1:GFRα1 pentameric interface was further validated both in vitro by native PAGE and in cellulo by cell-clustering and dendritic spine assays. Finally, we provide biochemical and cell-based evidence that RET and heparan sulfate cooperate to prevent assembly of the adhesion complex by competing for the adhesion interface. Our results provide a mechanistic framework to understand GDNF-driven cell adhesion, its relationship to trophic signalling, and the central role played by GFRα1.


SUPPLEMENTARY FIGURES
Supplementary Fig. 1.Purification, crystallographic and structural analysis of zGDNF mat -zGFRa1 D1-D3 multimers.(a) SEC profile of zGDNF mat -zGFRa1 D1-D3 run on a Superdex 200 10/300 column.SDS-PAGE gel of peak fractions is shown within the inset.(b) Self-rotation function (SRF) analysis of the 30 -5Å diffraction data from the zGDNF mat -zGFRa1 D1-D3 crystal.Left: The Chi = 180 o section of the SRF with a total of 10 peaks indicating ten 2-fold axes and Right: The Chi = 36° section of the SRF with a single peak indicating a 5-fold non-crystallographic symmetry axis.Self-rotation analysis was calculated and visualized with MOLREP in CCP4I 1,2 .(c) The two zGFRa1 pentamers from the zGDNF mat -zGFRa1 D1-D3 10:10 crystal structure are shown as a surface representation, with a single zGFRa1 protomer from each subunit coloured in grey.This highlights the 36° rotation around the 5-fold axis between the two sub-complexes.(d) Asparagine (N)-linked glycans within the zGDNF mat -zGFRa1 D2-D3 structure.Final electron density map (m2FO-DFc difference electron density map) contoured to 1s at each N-linked glycosylation site with the modelled Nlinked glycans superposed.The backbone of (i) zGFRa1 and (ii) zGDNF are shown in cartoon representation and the attached glycans as sticks.Schematics of modelled glycoforms were generated by PRIVATEER 3 .(e) SDS-PAGE analysis of proteolytic clipping time-course for the zGFRa1 D1 domain.Purified zGDNF mat -zGFRa1 D1-D3 was incubated for indicated timepoints at 30 °C and the extent of D1 clipping assessed by the generation of a new lower molecular weight band running at 25 kDa.(f) Structural superimposition of a single zGDNF mat -zGFRa1 D2-D3 1:1 assembly from the decameric complex (PDB: 8OS6) with the 1:1 zGDNF mat -zGFRa1 D2-D3 structure previously published (PDB: 7AB8) Conformational differences are seen in the relative angle of the helical structural element of GDNF, with a 15.4° rotation between the two helical elements.(g) Structural superimposition of a 2:2 zGDNF mat -zGFRa1 D2-D3 assembly from the decameric complex, and the 2:2 assembly of zGDNF mat -zGFRa1 D2-D3 previously published (PDB: 7AB8).Inter-protomer bend angles were calculated in PyMOL 4 by measuring the angle between two ß-finger elements and a central disulfide bridge, Glu162Ca-Cys202Sg-Glu162Ca' 5 .average cryo-EM map the zGDNF-zGFR⍺1 adhesion complex.Resolution reported based on the gold-standard FSC threshold (FSC = 0.143 criterion).

Supplementary
Supplementary Fig. 4. Membrane orientation and competing interfaces within the GDNF-GFRa1 adhesive complex and GDNF-GFRa1-RET trophic complex.(a) (i) zGDNF mat -zGFRa1 D2-D3+ decamer crystal structure (PDB: 8OS6) mediating adhesion between two opposing membranes.Shown as per Figure 1 with same domain colours.The distance between the membranes corresponds to 15 nm, as estimated from the reported 13.8 nm height of the zGDNF mat -zGFRa1 D2-D3+ decamer crystal structure.This contrasts with the width of the synaptic cleft reported to be 20 nm by electron microscopy of resin-embedded samples and around 25 nm in cryopreserved samples.This discrepancy is likely accounted for by the 65 amino acids from the C-terminal of each GFRa1 protomer (shown emanating from the D3 domain      and anchored to the cellular membrane via a GPI-modification) that would be present in the full-length but lacking in the GFRa1 D2-D3+ construct.ii) Cryo-EM map (EMD-11822) of the reconstituted zGDNF mat 2-zGFRa1 D1-D3 2-zRET ECM 2 complex segmented and coloured by protein chain as labelled.The schematic shows the different relative orientation of GDNF with respect to the cellular membrane and GFRa1 D2-D3 domains.(b) Sequence alignment of GFRa1 D2-D3 domains by Espript (http://espript.ibcp.fr) 9Secondary structure elements from the zebrafish GFRa1 D1-D3 structure (PDB: 7AML) are annotated above and disulphide linked cysteine pairs indicated by green numbers below.Invariant residues are boxed in red and similar residues in red text.Interaction residues at the GFRa1-RET high affinity site are boxed in pink and interaction residues at the GFRa1:GFRa1 pentamer interface are boxed in black.(c) Reducing SDS-PAGE gel of liposome pelleting membrane (P) and soluble fractions (S).His6-tagged zGFRa1 wild-type and mutant constructs conjugated to the liposome membrane are indicated above each lane and the presence/absence of untagged zGDNF mat and zRET ECM in each sample indicated (+/-).(d) Native-PAGE gel of zGDNF mat -zGFRa1 D2-D3+ in the presence or absence of zRET ECM as indicated.The dashed box indicates the position of the 700 kDa band corresponding to the decameric zGDNF-zGFRa1 D2-D3+ complex.The first lane is zRET ECM alone.In the presence of zRET ECM the formation of the zGDNF-zGFRa1 D2-D3+ decamer is disrupted in a Ca 2+ -dependent manner.Similar results were obtained in two other biological repeats.Both i & ii were stained using anti-HA antibodies (see Supplementary methods).(c) SPR analysis showing different mammalian GFRa1 mutants retain full GDNF binding functionality.i) Inset, hGDNF mat was covalently coupled to a CM5 chip and various concentrations of mGFRa1 D1-CT constructs were injected over the chip surface.The maximum response reached for each analyte concentration was fitted to a steady state 1:1 affinity binding model to determine an equilibrium binding constant (Kd) for each construct.ii) Table of determined Kd values for each construct representing the mean value of at least three technical replicates with the SE represented.N value indicates the number of technical repeats.(d) Analysis of expression and cellular localization of GFRa1 mutants in hippocampal neurons.Immunofluorescence imaging of hippocampal neurons co-transfected with GFRa1-HA mutants and GFP to show that they are expressed and localized correctly to the plasma membrane.Immunostaining was done with anti-HA antibodies (dil 1:400) on neuronal cultures that were not permeabilized.Nuclei were stained with DAPI.Scale bar: 25 µm.(e) Schematic representation of the synaptogenic assay using dissociated hippocampal neurons 10,11 , adapted from Paratcha and Ledda., 2008 12 .The consequences of mGFRa1 mutants presented on the postsynaptic membrane (transfected dissociated hippocampal neurons) was analysed by quantifying the dendritic spine density.The presynaptic membrane (axon) expresses endogenous mGFRa1 FL .Supplementary Fig. 6.SOS binding to GFRa1 is D1-dependent and GAGs do not impact preformed GFRa1-GDNF cell adhesion complexes.(a) ITC analysis of GFRa1 interactions with SOS.i) hGFRa1 D1-CT binding to SOS and ii) hGFRa1 D2-CT binding to SOS.Raw ITC titration data with the fitted offset subtracted plotted against time (top) and integrated heat signals plotted as a function of molar ratio (bottom).Circles represent the integrated heat of interaction, while blue curves represent the best fit obtained by non-linear least-squares procedures using the 'One set of sites'  5d) to probe the impact of sulfated GAGs on GFRa1 adhesion capacity in the presence of GDNF.HEK293T cells expressing mGFRa1 FL or mGFRa1 DD1 with GFP were preincubated with (i) SOS or (ii) HS both at 0.5 mg/ml for 2 h followed by 2 h of incubation with GDNF at room temperature.The control sample of transfected cells were not treated with GAG or GDNF.Scale bar :100 µm in (i) and : 50 µm in (ii) images.(c) HEK293 adhesion assay to probe the impact of GAGs on preformed GDNF-mGFRa1 assemblies within cell clusters.HEK293T cells were transfected with pCDNA3, mGFRa1 FL or mGFRa1 DD1 with GFP.GFP-expressing cells were then incubated with GDNF (as in Fig. 4b) for 2 h at room temperature.(i) SOS or (ii) HS (0.5 mg/ml) was added for an additional 2 h at room temperature Non-transfected HEK293T cells (a) forward vs side scatter (FSC vs SSC) area plot (red population) was analyzed in order to remove debris, followed by a side area vs side height (FSC-A vs FSC-H) to remove doublets.The tables show the percent of total HEK293T cells sorted including the doublets (HEK293T, in red), the percent of single HEK293T cells (Single Cells, in red), the percent of total GFP+ cells (GFP+ cells, in orange) and the percent of GFP+ cells used in the experiment (Experimental cells, in purple).Sorting settings were: nozzle of 85 micrón; precision: Purity; laser: 488 nm.

Expi293 Expi293
As above

Fig. 2 .
Biochemical and negative-stain EM validation of the zGDNF-zGFRa1 decameric assembly.(a) Reducing SDS-PAGE gel (i) and native-PAGE gel (ii) of purified zGDNF mat -zGFRa1 D1-D3+ .Purified zGDNF mat -zGFRa1 D1-D3+ was incubated for indicated timepoints at 30 °C and the extent of D1 clipping assessed -S E C u n c r o s s li n k e d C 1 c r o s s li n k e d e zGDNF mat -zGFRα1 D2-D3+

α 1 DSupplementary Fig. 3 .
by the generation of a new lower molecular weight band running at 40 kDa.By 192 h, almost complete cleavage of the D1 domain has occurred.A high molecular weight band (~700kDa), indicated by the red box, consistent with the decameric complex, can be observed at 192 h by native-PAGE formed from a 2:2 complex in solution upon proteolytic clipping of the N-terminal D1 domain.(b) Reducing Tris-Acetate SDS-PAGE gel of crosslinked recombinant zGDNF mat -zGFRa1 samples.zGFRa1 recombinant samples in complex with zGDNF mat indicated above the gel.The red box indicates a higher molecular weight band > 225 kDa observed for zGDNF mat -zGFRa1 D2-D3+ sample.(c) SEC profile of crosslinked zGDNF mat -zGFRa1 D2-D3+ and reducing SDS-PAGE gel of fractions across the elution peak.The red dashed box indicates the gel-filtration fraction, C1, applied to EM grids and negatively stained (NS).(d) Representative NS-EM micrograph of purified crosslinked zGDNF mat -zGFRa1 D2- D3+ , from a total of 540 micrographs (e) Projection matching performed using xmipp3 "compare reprojection" protocol 6 between the RELION (v3.1) 7,8 2D class averages from the particles used to generate the final 3D reconstruction, and different reprojections of the 3D model.(f) Fourier shell correlation (FSC) curve of the zGDNF mat -zGFRa1 D1-D3+ NS-EM map.Resolution reported based on the goldstandard FSC threshold, FSC = 0.143, shown as a dashed line.(g) Native-PAGE gel of zGDNF mat -zGFRa1 D2-D3+ prior to crosslinking (left-hand lane) and post crosslinking and SEC purification (right-hand lane).(h) The orientation distribution of particles that contribute to the final reconstruction of the zGDNF mat -zGFRa1 D2-D3+ decameric complex.The predominant view is parallel to the five-fold molecular dyad (side view), and a fraction of particles viewed perpendicular to the 5-fold rotational symmetry (top view).Visualising uncrosslinked GDNF-GFRa1 adhesion complexes on reconstituted liposomes by cryo-ET.(a) Dynamic light scattering size distribution analysis of liposomes prepared using DOPC:DGS-Ni 2+ -NTA lipids.Shown is a plot of the determined intensity-weighted mean hydrodynamic particle size distribution of DOPC:DGS-Ni 2+ -NTA liposomes revealing an average liposome diameter of 129.9 d.nm.d.nm = particle diameter in nm.(b) 2D tomographic slices from binned by 4 reconstructed tomograms of zGDNF mat -zGFRa1 D2-D3+ -anchored liposomes.Images show close-up views of bridging protein density between two liposome membranes (indicated with white arrow heads).Two views project down the assembly 5-fold axis.Middle left and bottom right panels.Scale bar: 20 nm.(c) Processing pipeline for reconstructing tomograms of zGDNF-zGFR⍺1 adhering liposomes and sub-tomogram averaging of the zGDNF-zGFR⍺1 adhesion complex.Particles belonging to the highest resolution class with good quality density (indicated by a black box) were selected during ab initio model generation and 3D classification.Numbers below each map indicate the total number of particles contributing to each 3D class average.A map calculated with C1 symmetry indicating the assembly exhibited 5-fold symmetry, therefore the final map was reconstructed with D5 symmetry imposed (d) Fourier shell correlation (FSC) curve of the final sub-tomogram Ab initio model generation (RELION v4.0 R S P N A I E P A T H I N H L N A D N S L Y Q F G K .D V D P Q I D D P S S S N T K N S S . . . .P R Q M T L S G L S Q L L T S H C I F T P D P Q V E H T R G T N T I G R D D S S A L . . . . . . . ... S L C T L T I T T Q S L A I G K D N T P G T S H I S E N S F A L P T S F Y P S T P L T I L S L F F N Y E K E G L .G S .H I T K S M A A P P S C G L P L L .V T L S T L S D Y G K D G L A G S .H I T K S M A A P P S C G L S L P .F T L A A L S D Y E K D G L A G S .H I T K S M A A P P S C G L P L L .V T L S T L S R S P N A I E P A T H I N H L N A D N S L Y Q F .D V D P Q I D D P S S S N T K N S S . . . .P R Q M T L S G L S Q L L T S H C I F T P D P Q V E H T R G T N T I G R D D S S A L . . . . . . . ... S L C T L T I T T Q S L A I G K D N T P G T S H I S E N S F A L P T S F Y P S T P L T I L S L F F N Y E K E G L .G S .H I T K S M A A P P S C G L P L L .V T L S T L S D Y G K D G L A G S .H I T K S M A A P P S C G L S L P .F T L A A L S D Y E K D G L A G S .H I T K S M A A P P S C G L P L L .V T L S T L S aggregates of 5 or more cells/field.Mean values of triplicate experiments (each measured in duplicate) ± s.e.m. were assessed by one-way analysis of variance (ANOVA), followed by Tukey´s multiple comparison test.****p < 0.0001 (b) Analysis of expression and cellular localization of HA-tagged mGFRa1 mutants in HEK293T cells.i) Immunofluorescence imaging of HEK293T cells transfected with mGFRa1 mutants demonstrate that mutants are correctly localized at the plasma membrane.Scale bar: 10 µm.ii) Immunoblot to show the level of expression of indicated mGFRa1 variants compared to wild-type in HEK293T cells.
titrations and binding curves of triplicate measurements are shown.Derived binding constants (Kd) are reported on each plot, as mean values of 3 independent experiments ± standard deviation.(b) Confocal images of HEK293 adhesion experiment (Fig. . The percentage of cells in aggregates greater than 5 cells under the indicated conditions is shown.Mean values of triplicate experiments (each measured in duplicated) ± s.e.m.One-way ANOVA, followed by Tukey´s multiple comparison test.The dashed lined indicates the percentage of cell aggregates in the absence of GDNF.ns indicates no statistically significant difference.Supplementary Fig. 8 Flow cytometry strategy for cell adhesion assay.Sorting strategy for live-cells control (a) and transfected cells (b) using the FACS Aria-Fusion and the FACsDiva versión 8.0.2 software.FSC Area scaling was adjust at 0.45.All live cells were sorted to collect the GFP + population (purple dots in the graphs).